Gene therapy for the X-linked bleeding disorder hemophilia (which is caused by mutations in the gene encoding blood coagulation factor VIII, hemophilia A, or factor IX, hemophilia B) has been tested in several Phase I/II clinical trials in recent years. Clinical protocols included muscle- and liver-directed factor IX (F.IX) gene transfer with adeno-associated viral (AAV) vectors. Data from these trials and from pre-clinical studies in animal models indicate that immunological responses likely represent the largest hurdle for successful therapy by this otherwise safe, efficacious, and highly promising treatment approach. Equally important, formation of inhibitory antibodies (inhibitors) to coagulation factors is a general concern and a serious complication of any treatment of hemophilia. Maintaining unresponsiveness of the immune system to the transgene product is a key requirement for successful gene therapy. In gene replacement therapy, the therapeutic gene product may represent a neo-antigen that can be subject to an adaptive immune response. Inhibitor formation to F.IX is an example of such a response, which is typically dependent on CD4+ T help. The most desirable outcome of treatment by gene transfer is induction of immune tolerance to the therapeutic protein mediated by transgene expression without a need for additional manipulation of the immune system. In recent studies, we demonstrated that a minimal level of expression upon hepatic gene transfer with AAV-2 vectors reliably induced robust immune tolerance to F.IX in several but not all strains of mice. The goals of this application are to establish an optimal protocol for induction of immune tolerance to F.IX by hepatic gene transfer, to investigate the mechanism of how hepatic antigen presentation leads to T cell tolerance to the transgene product, and to develop protocols for in vivo generation of F.IX-specific regulatory T cells. We hypothesize that an optimal hepatic gene transfer protocol can achieve a high success rate of tolerance induction regardless of the genetic background of the animal receiving gene transfer. Immune tolerance will be defined as sustained hepatic and systemic F.IX expression (without antibody formation) and failure to respond to subsequent immunization with F.IX protein. An alternative strategy to prevent deleterious immune responses to the transgene product is the generation of F.IX-specific regulatory T cells prior to gene transfer. Based on the hypothesis that efficient activation of regulatory T cells prevents F.IX-specific T and B cells during subsequent therapy, two alternative strategies will be evaluated: mucosal (nasal) antigen administration and a novel protocol designed to delete effector T cells concomitant with induction of CD4+CD25+ regulatory T cells. All strategies will be compared for duration and robustness of unresponsiveness to F.IX. Public Health Relevance Statement: This project will develop novel strategies for prevention of undesired immune responses in treatment of the inherited bleeding disorder hemophilia. Immune responses to coagulation factors represent a severe complication of treatment of patients with hemophilia, increase morbidity and mortality, and substantially increase cost of treatment. Therefore, superior immune tolerance protocols are highly desirable.